Graft versus host disease (GVHD) is the major complication associated with allogeneic bone marrow transplantation (BMT). The progression of inflammatory events that results in widespread tissue damage develops from the imbalance between the effector and regulatory arms of the immune system. In particular, the sustained and progressive decrease in regulatory T cells (Tregs) is a hall mark of GVHD and unleashes effector T cells to mediate direct cytotoxic effects as well as promote the secretion of inflammatory cytokines. The re-establishment of effective Treg control of GVHD through adoptive transfer or in vivo augmentation is an area of intense interest and has significant clinical implications. However, Tregs represent a heterogeneous population and there remain numerous questions with respect to the specific regulatory T cell populations that contribute to the maintenance of tolerance in GVHD biology. The overall goal of this proposal is to define the role of specific regulatory T cell populations in the pathophysiology of GVHD in order to develop clinically based strategies that will facilitate transplantation tolerance. Our overall hypothesis s that Treg populations have unique roles in GVHD biology and that the cooperative function of these populations is necessary for optimal GVHD protection after allogeneic stem cell transplantation. To address this hypothesis, experiments have been designed to address the following specific aims. Studies in Specific Aim 1 will define the role of in vivo-induced CD4+ Tregs (iTregs) in mitigating GVHD and to determine the extent to which these cells complement CD4+ natural Tregs (nTregs) in the prevention of this disease. We will also define the role of in vitro-differentiated CD4+ iTregs in preventing GVHD. We will exploit the ability to stabilize Foxp3 expression in in vitro-generated CD4+ Tregs using clinically relevant pharmacological approaches and determine how these cells cooperatively function with CD4+ nTregs for GVHD prevention. Finally, studies will be conducted to define the role of interleukin 10 in mediating th suppressive effects of CD4+ in vivo-induced iTregs, in vitro-differentiated iTregs, and nTregs. Specific Aim 2 will examine the role of a novel CD8+ Foxp3+ Treg population that we have identified and found to suppress GVHD. We will define the role of in vivo and in vitro-generated CD8+ Tregs in the prevention of GVHD, the temporal requirement for these cells to exert suppressive effects, and the extent to which the generation of these cells is modulated by Stat 3 dependent cytokines. We will also determine the T cell repertoire and transcriptional signature of these cells in comparison to natural and induced CD4+ Tregs, and the mechanisms by which these cells suppress alloresponses. The overall objective of this proposal is to develop clinically based strategies that will facilitate transplantation tolerance, reduce GVHD-associated mortality, and improve overall survival.